.alpha..sub.1 -Antitrypsin (.alpha..sub.1 -AT), .alpha..sub.2 -Macroglobulin (.alpha..sub.2 M) and .alpha..sub.1 -antichymotrypsin account for about 90% by weight of the total plasma protease inhibitors. The three inhibitors are high molecular weight glycoproteins and are capable of inhibiting various proteolytic enzymes containing active-site serine residues. They act by allowing their target proteases to bind directly and irreversibly to a substrate-like region within the amino acid sequence of the inhibitor. In almost all cases, the resulting complex is of 1:1 stoichiometry i.e. one molecule of each reactant. This property forms the basis of a variety of serum protease inhibitor capacity assays utilizing trypsin or chymotrypsin as proteases (J. H. Eckfeldt et. al., Clin. Chem. 1982, vol. 28, pp 1108-1112 and J. Wenger and M. Sundy, Clin. Chem. 1974, vol. 20, pp 328-331). In most tryspin or chymotrypsin inhibitor capacity assays it has been shown that .alpha..sub.1 -AT is invariably measured (F. Miesch et al. Clin. Chem. Acta. 1971 vol. 31 pp 231 and above mentioned references).
.alpha..sub.1 -Antitrypsin, due to its functional relationship in various disease states, is subject of importance at clinical diagnostic level. Deficiency of .alpha..sub.1 -AT can arise due to specific genetic defects in certain individuals. This leads to a lung disease, familial emphysema, at an early age (S. Erikson, Acta. Med. Scand., 1965, vol. 177, suppl: 432), liver disease (N. Berg and Erikson; N.E.J. Med., 1972, vol. 287, pp 1264-1267). About 5 to 20% of infants with inherited .alpha..sub.1 -AT deficiency suffer from infantile liver cirrhosis leading to fatal hepatic failure or hemorrhage during childhood (H. L. Sharp; Gastro, 1970, vol. 70, pp 611621).
The most common (nongenetic) change is due to the acute phase response where .alpha..sub.1 -AT levels are elevated in most conditions associated with infection, inflammation or tissue necrosis (J. O. Jeppsson, C. B. Laurell and B. Franzen, Clin. Chem 1979, vol. 25; pp 629-638). Elevation is rapid, within 24 hours of the stimulus, and continues for as long as the stimulus is present. Increased .alpha..sub.1 -AT synthesis is also seen with pregnancy, oestrogen therapy and some forms of hepatocellular disease. .alpha..sub.1 -AT levels are reduced in association with neonatal respiratory distress syndrome.
Overall, the major clinical interest is .alpha..sub.1 -AT deficiency, especially where neonatal screening can detect the genetically based hepatic diseases before becoming clinically apparent. There is also a role in the detection of heterozygotes where genetic counselling is indicated. .alpha..sub.1 -AT measurement is useful for monitoring acute phase responses and there may be a role for .alpha..sub.1 -AT measurement in the prediction and monitoring of neonatal idiopathic respiratory distress syndromes.
Despite its importance, the measurement of protease inhibitor capacity has not found widespread use because most assays are manual, laborious and, in a number of cases, employ complex and expensive instrumentation. In addition, the lack of absolute standardization in the current assays is a serious problem and prevents meaningful interlaboratory comparison. Most of these assays are based on the inhibition of trypsin or chymotrypsin and the results are expressed as inhibition either as per mass of enzyme or as per units of enzyme activity inhibited by a given volume of serum. The use of mass is, however, incorrect because commercially available crystalline enzymes are not 100% enzymically active and .alpha..sub.1 -AT reacts with only active enzyme fraction. The operational molarity of these enzymes could easily vary between 40 to 70% of the mass as shown by active-site titration techniques (T. Chase and E. Shaw; Biochem. Biophys. Res. Commun., 1967, vol. 29, pp 508-514 and B. E. Erlanger and R. A. Sack; Anal. Biochem., 1970, vol. 33, pp 318-322). The use of the enzyme units inhibited per volume of serum also has drawbacks. The substrates most commonly used have limited solubility and enzymes are operating at nonsaturating substrate concentration. This condition, commonly referred to as non zero-order kinetics, makes the estimation of enzyme activity very sensitive to small variations in substrate concentration.
Immunochemical techniques offer the advantage of high specificity but these techniques do not distinguish enzymically active complexes from inactive complexes (J. A. Pierce; New England J. Med. 1972, vol. 287, pp 1905 and L. Gaidulis et. al., Clin. Chem., 1983, vol. 29, pp 1838-1840).
Most of the current assays are furthermore based on photometric techniques, which, beside requiring expensive intrumentation, work satisfactorily only with test solutions of certain optical purity. Thus highly turbid, lipemic or icteric serum can be problemetic in these assays.
The main aim of the present invention is to provide an improved method for the assay of protease inhibitor capacity of serum. A further aim of the present invention is to provide a method which can unambiguously measure serum protease inhibitor capacity by using an active-site titration technique independent of test protease enzyme supplier, and which does not require optically pure test solution. A still further aim of the present invention is to provide means to perform these assays with inexpensive instrumentation and which may be readily automated.
Assays for serum protease inhibitor capacity are generally based on the stoichiometric 1:1 combination of the inhibitor with an excess and known amount of trypsin or chymotrypsin. Since the resulting complex is enzymically inactive, the remaining active fraction of the enzyme provides a measure of serum inhibitor capacity. It has now been found that the serum inhibitor capacity assay is improved and greatly simplified by employing a specific active-site titrant for the direct determination of the enzyme. Active-site titrants are an art recognized class of enzyme reagents and are frequently employed in the determination of the "operational concentration" of enzymes. An active-site titrant is a nonprotein substance and a specific inactivator of a given enzyme. The titrant reacts mole for mole with some amino acid residues in the active-site of the enzyme. The reaction principle may be represented by the following equation. EQU RL+enzyme.fwdarw.R-Enzyme+L*
where RL is the active-site titrant, R-Enzyme is the titrated inactive enzyme complex and L* is the product released which, depending on its nature, can be measured by a number of available sensitive techniques. In the case of serum protease inhibitor capacity assays this principle has recently been exploited using photometric detection technique (J. H. Eckfeldt et. al., Clin. Chem. 1982, vol. 28, pp. 1108-1112). Although this publication clearly shows the advantage of active-site titration, the requirement of expensive instrumentation is its serious drawback. The present invention utilizes the known phenomenon that diphenylcarbamyl fluoride (DPCF) is a specific inactivator of .alpha..sub.1 -chymotrypsin and trypsin and can be used as active-site titrant. The DPCF reaction is stoichiometric with the release of F.sup.- ion which then can be measured by a fluoride ion-selective electrode (B. F. Erlanger and R. A. Sack, Anal. Biochem., 1970, vol. 33. pp 318-322).
We have found that the technique used for active-site titration as described in the above mentioned publication can not be used for the assay of serum protease inhibitor capacity for the following reasons:
(i) The active-site titration requires at least 30 minutes of incubation prior to F.sup.- measurement which is somewhat inconvenient if a large number of serum samples are to be processed as in screening studies. PA0 (ii) The lack of constant ionic strength buffer can be a serious source of error in F.sup.- measurement by the fluoride ion-selective electrode. PA0 (iii) In actual serum protease inhibitor capacity assays, the 30 minute incubation with DPCF for active-site titration of chymotrypsin results in anamalous F.sup.- ion generation probably due to some unexplained reactions with the test serum components.